1. Field of the Invention
The present invention relates generally to the field of oligonucleotide chemistry and anti-viral pharmacotherapy. More specifically, the present invention relates to therapeutically active guanosine-rich intramolecular tetrad forming oligonucleotides, to methods of treating viral diseases using said oligonucleotides, and to pharmaceutical compositions containing the novel oligonucleotides.
2. Description of the Related Art
General In Vitro Studies
Previously, it was believed that "antisense" oligonucleotides inhibit viruses by interfering with protein translation via an RNA:DNA duplex structure. More recent research, however, indicates a variety of possible mechanisms by which oligo-nucleotides inhibit viral infections. For example, oligodeoxycytidine (poly SdC) inhibits HIV-1. Marshall et al., PNAS (1992) 89:6265-6269, discussed the potential mechanism (competitive inhibition) by which oligodeoxycytidine directly inhibits viral reverse transcriptase. Poly SdC also inhibited AMV reverse transcriptase and Pol I (Klenow fragment) and polymerase .alpha., .beta. and .gamma.. Previously, Matsukura et al., PNAS (1987) 84:7706-7710, used a similar phosphorothioate derivative of oligo-deoxycytidine to demonstrate inhibition of HIV-1 in culture. Marshall and Caruthers, Science (1993) 259:1564-1569, reported the use of diphosphorothioate oligo-nucleotides, e.g., antisense-specific, random nucleotide combinations and oligodeoxycytidine against HIV-1. In all cases, the mechanism of action was attributed to a direct inhibition of HIV-1 reverse transcriptase. Other potential mechanisms of anti-viral action of oligonucleotides were postulated by Boiziau et al., PNAS (1992) 89:768-772, e.g., promotion of RNAse H activity and inhibition of reverse transcriptase initiating cDNA synthesis. In addition, Goa et al., Molecular Pharmacology (1992) 41:223-229 reported that phosphorothioate oligonucleotides inhibit human DNA polymerases and RNAse H, and the adsorption or penetration of the virus into cells. Iyer et al., Nucleic Acids Research (1990) 18:2855-2859 reported that if a base was removed from an anti-sense polynucleotide forming an abasic site, the compound did not lose its activity which argues against the need for the formation of an RNA:DNA antisense mediated hybrid for anti-viral activity. Stein et al. have characterized the interaction of poly SdC with the V3 loop of HIV-1 gp120, and postulated that the specific interaction of poly SdC with the HIV-1 V3 loop may be a mechanism by which an oligonucleotide could inhibit HIV-1 in vivo.
It is known that synthetic oligonucleotides may be designed which are capable of binding to duplex DNA to form triplex DNA. See U.S. Pat. No. 5,176,996 Hogan & Kessler issued Jan. 5, 1993. That patent discloses a method for making synthetic guanosine-rich oligonucleotides which are targeted to specific sequences in duplex DNA and which form collinear triplexes by binding to the major groove of the DNA duplex.
Specific In Vitro Studies/In Vitro HIV Inhibition With T30177
Infection with the human immunodeficiency virus type 1 (HIV-1) and the subsequent development of acquired immunodeficiency syndrome (AIDS), has become a threat to public health on a global scale. Preventing further spread of this disease is a major health priority worldwide. Although HIV-1 was confirmed to be the causative agent of AIDS as early as 1984, few drugs and no vaccines are effective at preventing the ultimate onset of AIDS in HIV-1 seropositive individuals. This is due, in large part, to the complexity of the causative agent itself, the dynamics of virus production and the speed at which drug-resistant mutants can arise. Ho, et al., Nature 373:123-126 (1995); Wei, et al., Nature 373:117-122 (1995).
Infection of T-cells by HIV-1 results in the insertion of proviral (double-stranded) DNA into the host cell genome. Goff, S. P., Annu. Rev. Genet. 26:527-544 (1992). The integration process involves both the sequence-specific and sequence independent endonucleolytic and strand transfer activities of the virally encoded integrase enzyme. Katz, et al., Ann. Rev. Biochem. 63:133-173 (1994); Vink, et a., Trends in Genetics 9:433-438 (1993). Once the proviral state is established, the infection may manifest itself in several ways including a latent infection in which viral replication is not measurable until the cell becomes activated or through a chronic infection in which dividing or non-dividing cells persistently release virus in the absence of any cytopathic effect. In addition, recent reports on the kinetics of virus production (and clearance) indicate a dynamic process in which virtually a complete replacement of wild-type virus by drug-resistant virus in plasma can occur after only two to four weeks of drug therapy. Ho, et al., Nature 373:123-126 (1995); Wei, et al., Nature 373:117-122 (1995). For this reason it is of utmost importance to develop new anti-HIV-1 agents which can complement, by additive or synergistic activity, current therapies.
One relatively new approach used in the development of antiviral therapeutics for HIV-1 is the use of oligonucleotides designed as antisense agents. Letsinger, et al., Proc. Natl. Acad. Sci. USA 86:6553-6556 (1989); Lisziewicz, et al., Proc. Natl. Acad. Sci. USA 90:3860-3864 (1993); Milligan, et al., J. Med. Chem. 36:1923-1937 (1993). While much effort is being spent on rationally designed oligonucleotides such as antisense agents there have also been recent findings of multiple alternative mechanisms by which oligonucleotides can inhibit viral infections. Gao, et al., J. B. C. 264:11521-11526 (1989); Marshall, et al., Proc. Natl. Acad. Sci. USA 89:6265-6269 (1992); Ojwang, et al., J. AIDS 7:560-570 (1994); Rando, et al, J. Biol. Chem. 270:1754-1760 (1995). For example, Stein et al. (Stein, et al., Antisense Research and Development 3:19-31 (1993)) have characterized the interaction of oligodeoxycytidine, containing a phosphorothioate (PT) backbone (poly (SdC)) with the V3 loop of HIV-1 gp 120. It was determined that poly (SdC).sub.28 specifically interacted with the positively charged V3 loop with a Kd of approximately 5.times.10.sup.-7 M. Stein et al. (Antisense Research and Development 3:19-31 (1993)) then postulated that the interaction of poly (SdC) with the HIV-1 V3 loop may be a mechanism by which poly (SdC) could inhibit HIV-1 in vivo. More recently, Wyatt et. al. (Wyatt, et al., Proc. Natl. Acad. Sci. USA 91:1356-1360 (1994)) have described the interaction of a short G-rich oligonucleotide, synthesized with a total PT backbone, which also interacts with the v3 loop of HIV-1 gp 120. In addition, we have previously reported that oligonucleotides containing only deoxyguanosine (G) and thymidine (T), synthesized with natural phosphodiester (PD) internucleoside linkages, were capable of inhibiting HIV-1 in culture. Ojwang, et al., J. AIDS 7:560-570 (1994). The most efficacious member of this dG-rich class of oligonucleotides, I100-15, was found capable of folding upon itself to form a structure stabilized by the formation of two stacked guanosine-tetrads which yielded a guanosine-octet. Rando, et al, J. Biol. Chem. 270:1754-1760 (1995). Furthermore, it was observed that the positions of the guanosine bases in the I100-15 sequence, found in both the tetrads and connecting loops in that structure, were extremely important to the overall anti-HIV-1 activity of the oligonucleotide. Rando, et al, J. Biol. Chem. 270:1754-1760 (1995).
Site of Activity Studies-Viral Integrase Inhibition
Two events which are characteristic of the life cycle of retroviruses can be utilized for therapeutic intervention. One is reverse transcription, whereby the single-stranded RNA genome of the retrovirus is reverse transcribed into singled-stranded cDNA and then copied into double-stranded DNA. The next event is integration, whereby the double-stranded viral DNA generated by reverse transcriptase is inserted into a chromosome of the host cell, establishing the proviral state. Integration is catalyzed by the retroviral enzyme integrase which is encoded at the 3'-end of the pol gene. Varmus, et al. Mobile DNA, pp. 53-108, Am. Soc. Microbiol, Washington, D.C. (1989). Integrase first catalyzes the excision of the last two nucleotides from each 3'-end of the linear viral DNA, leaving the terminal conserved dinucleotide CA-3'-OH at these recessed 3' ends (FIG. 23A). This activity is referred to as the 3'-processing or dinucleotide cleavage. After transport to the nucleus as a nucleoprotein complex, Varmus, et al. Mobile DNA, pp. 53-108, Am. Soc. Microbiol, Washington, D.C. (1989), integrase catalyzes a concerted DNA strand transfer reaction by nucleophilic attack of the two viral ends onto a host chromosome. This reaction generates a recombination intermediate resembling an X structure, analogous to a Holliday junction intermediate. [For recent reviews see Katz and Skalka, Katz, et al., Ann. Rev. Biochem. 63, 133-173 (1994), and Vink and Plasterk, Vink, et al., Trends Genet. 9, 433-437 (1993)]. Mutation analyses of the viral integrase gene demonstrate that integration is required for effective retroviral replication and that it is a legitimate target for the design of antiretroviral drugs (Engleman, et al., J. Virol. 69, 2729-2736 (1995); Englund, et al, J. Virol. 69, 3216-3218 (1995)).
It is known that AZT nucleotides can inhibit HIV-1 integrase, Mazumder, et al., Proc. Natl. Acad. Sci. 91, 5771-5775 (1994), and that substitution or unsaturation at the 3'-position of the deoxyribose confers potency against HIV-1 integrase. These results suggested that the enzyme's nucleotide binding site could serve as a potential drug target. It has been shown that the potential stacking interactions gained from the heterocyclic rings can further enhance potency against HIV-1 integrase.
Recently, oligonucleotides composed of deoxyguanosine and thymidine have been reported to inhibit HIV-1 replication. Rando, et al., J. Biol. Chem. 270, 1754-1760 (1995); Wyatt, et al., Proc. Natl. Acad. Sci. U.S.A. 91, 1356-1360 (1994). Oligonucleotides forming intramolecular G4s did not block virus adsorption but rather inhibited viral-specific transcripts. Rando, et al., J. Biol. Chem. 270, 1754-1760 (1995); Ojwang et al. J. Aids 7:560-570 (1994).
Structure-Function Studies
It is known that G-rich nucleic acid sequences can fold, in the presence of Na.sup.+ or K.sup.+ ion, to form orderly structures stabilized by guanosine tetrads. Depending on sequence, intramolecular folds, Rando et al. J. Biol. Chem. 270:1754-1760, 1995), dimers (Smith, F. W., & Feigon, J. (1992) Nature (London) 344, 410-414, Sundquist, W. I. & Klug, A. (1989) Nature (London) 334, 364-366; Kang, et al. (1992) Nature (London) 356, 126131; Balaguumoorthy, P. & Brahmachari, S. K. (1994) J. Biol. Chem. 269, 21858-21869), tetrameres (Son, D. & Gilbert, W. (1990) Nature (London) 344, 410-414; Jin, et al. (1990) Science 250, 543-546; Jin, et al. (1992) Proc. Natl. Acad. Sci. USA 89, 8832-8836; Lu et al., (1992) Biochemistry 31, 2455-2459), and higher order associations have been detected. Such tetrad based structures have been postulated to serve as the structural basis for telomere function (Sen, D. & Gilbert, W. (1988) Nature (London) 334, 364-366), and have been hypothesized to play a role in retroviral replication (Bock et al. (1992) Nature (London) 355, 564-566), and transcription regulation (Marshall et al. (1992) Proc. Nalt. Acid. Sci. USA 8,9, 6265-6269; Wyatt et al. (1994) Proc. Natl. Acad. Sci. USA 91, 1356-60).
Recently, several groups have shown that compounds which contain tetrad-based folds may have activity as potential drug compounds. Bock and colleagues have shown that an intramolecular fold, obtained by a SELEX procedure can bind tightly to thrombin, so as to inhibit clotting (Bock et al. (1992) Nature (London) 355, 564-566). Additionally Wyatt et al. (Wyatt et al. (1994) Proc. Natl. Acad. Sci. USA 91, 1356-60) has shown that a dimer-wise pairing of phosphorothioate oligomers with the sequence T2G4T2 (four stranded intermolecular tetrads) gives rise to anti-HIV activity, by inhibition of viral adsorption to the cell surface.
The present inventors have also obtained evidence for sequence-selective inhibition of HIV-1 by simple phosphodiester oligonucleotides which form G-tetrad based structures. The highest activity was obtained with a 17mer, referred to as T30177, with composition G12-T5 (Rando et al., (1994) J. Biol. Chem. 270, 1754-1760; Ojwang, J. et al. (1995) J. Aids 7, 560-570), with 2 phosphorothioate linkages (1 at each end) to block cellular exonuclease activity (Bishop et al. (1996) J. Biol. Chem. 271, 5698-5703). NMR evidence was obtained (Rando et al., (1995) J. Biol. Chem. 270, 1754-1760) to suggest that, by reference to similar oligomers (Smith, F. W., & Feigon, J. (1992) Nature (London) 344, 410-414), T30177 forms a stable intramolecular fold which is stabilized by a pair of G-tetrads, connected by three single-stranded loops and a 1-2 base long tail to either side of the fold. Those preliminary studies suggested that oligomer folding was coupled to K.sup.+ ion binding (Rando et al., (1995) J. Biol. Chem. 270, 1754-1760). Additional studies have suggested that T30177 and related derivatives are potent inhibitors of HIV-1 integrase, in vitro (Ojwang et al. (1995) Antimicrob. Agent Chemotherepy 39, 2426-35).
Pharmacokinetic Studies-Single Dose
Antisense, triple-helix, duplex decoy, and protein-binding (aptamer) oligonucleotides have been shown to have potential as drugs for the treatment of a variety of human clinical disorders (Stein and Cheng, 1993; Marshall and Caruthers, 1993, Science 259:1564-1570; Chubb and Hogan, 1992, Trends in Biotechnology 10:132-136; Stull and Szoka, 1995, Pharm. Res. 12:465-483. A number of oligonucleotides have undergone pre-clinical testing, and several are in human clinical trials. One finding that has aroused some concern (Black et al., 1994, Antisense Res. Dev. 4:299-301) is the observation that total phosphorothioate oligonucleotides cause hemodynamic changes following rapid intravenous administration. Severe hypotension, leukopenia, complement activation, and death have been reported to occur in primates after rapid infusions of total phosphorothioate oligonucleotides (Cornish et al., 1993, Pharmacol. Commun. 3:239-247; Galbraith et al., 1994, Antisense Res. Dev. 4:201-206). These findings have raised the question of whether the cardiovascular toxicity is a property of phosphorothioate oligonucleotides, or of all oligonucleotides. On the basis of these findings, an FDA commentary has recommended that cardiovascular screening be performed for the pre-clinical safety assessment of oligonucleotides (Black et al., 1994).
Pharmacokinetic Studies-Repeat Dose
Oligonucleotides have advanced to the stage that they are now considered as potential therapeutics for the treatment of a variety of human diseases, and several are presently in clinical trials. Pre-clinical studies have generally shown that doses up to approximately 50 mg/kg are safe, but that higher doses can cause kidney and liver damage, and death (Srinivasan and Iversen, 1995, J. Clin. Lab. Analysis 9:129-137) Bolus intravenous administration has posed a particular concern since it has been shown to sometimes result in serious hypotensive events in primates (Cornish et al., 1993; Galbraith et al., 1994; Black et al., 1995). However, because the number of oligonucleotides that have been studied has been small, it is difficult to conclude at the time of making the invention whether all oligonucleotides share similar toxicities. In particular, given the various ways of modifying the backbone of oligonucleotides (Wu-Pong, 1994, BioPharm 7:20-33) and their ability to fold into distinct three-dimensional structures (Stull and Szoka, 1995, Pharm. Res. 12:465-483), Rando et al. J. Biol. Chem. 270; 1754-1760, 1995, the safety profile of different oligonucleotides may be quite distinct.
Human Clinical Trials
In addition to toxicological studies, efficacy studies should be carried out for oligonucleotide drugs. In the past, the preferred method of testing drug efficacy, especially in HIV-1 infected patients, was to monitor survival of treated patients. However, recent statistical studies have shown that a good indicator of anti-HIV drug efficacy is the reduction in the numbers of copies of viral genome per unit of patient serum (viral load). Mellors et al. (1996) Science 272:1167-1170. Reductions in viral load of 90%, or more preferably 99% are desired. However, reductions of viral load of lesser percentages can be useful, especially where the trend of the overall treatment regime is consistently downward.
Thus, there is a substantial need for antiviral drugs with novel chemistry and with sites of activity distinct from drugs presently used. Most highly desired would be antiviral drugs whose efficacy in humans is known.